Atherosclerosis, a progressive chronic inflammatory disease of the vessel wall, is regulated by oxidant stress throughout the course of disease development. Endothelial dysfunction is a critical, initiating step in the development of atherosclerosis and increasing evidence implicates mitochondrial reactive oxygen species (ROS) as an important contributor to endothelial dysfunction and vascular inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is emerging as an important enzyme in the regulation of mitochondrial NADPH levels which can have a significant impact on a number of metabolic pathways through the regulation of mitochondrial redox tone (balance of ROS production and removal). Our preliminary data supports this concept and indicates that that the absence of NNT in C57Bl/6J (6J) cells led to distinct mitochondrial bioenergetic profiles and a pro-oxidative mitochondrial phenotype characterized by increased superoxide production and reduced glutathione peroxidase activity. Interestingly, we found that 6J animals are more susceptible to high fat diet induced plaque formation compared to C57Bl/6N (6N). Plaque formation was driven by increased plasma lipids in both animals. However, treatment with the mitochondria targeted superoxide dismutase mimetic MitoTEMPO had distinct effects on serum lipids and plaque formation in the 6N and 6J animals. MitoTEMPO treatment reduced plasma lipids in the 6N animals but not the 6J, and surprisingly, exacerbated plaque formation in the 6J animals, demonstrating a critical role for the production of mitochondrial reactive oxygen species in the development of atherosclerosis in these animals. Building upon these findings, we propose that the loss of NNT activity contributes to a pro-oxidative mitochondrial phenotype that exacerbates the progression of atheroscelrosis by enhancing mitochondrial ROS production, endothelial dysfunction, and vascular inflammation. To test this hypothesis, studies are proposed that will determine i) if NNT inhibits mitochondrial ROS production and preserves normal endothelial ?NO function in human vascular endothelial cells; ii) if reduced NNT expression will increase mitochondrial ROS production and stimulate Nox activity that contributes to endothelial dysfunction; and iii) if endothelial NNT critically regulates mitochondrial redox tone and vascular function in mice treated with high fat diet. Data from the proposed studies will identify NNT as a master regulator of mitochondrial function and ROS production whose absence exacerbates the development of atherosclerosis by promoting endothelial dysfunction and vascular inflammation, leading to increased plaque development.